We propose to examine the interrelationships between the cyclic AMP system and retinoic acid (Vitamin A acid) in the regulation of cell growth and differentiation. Specifically, we will study the manner by which retinoic acid enhances protein kinase activity in B16 melanoma cells by partially purifying and characterizing the protein kinase from control and retinoic acid treated cells. Direct quantitation of the amount of regulatory subunit of protein kinase in control and treated cells will be achieved by use of the photo-affinity analog 8-azido-cyclic AMP32. Variants of B16 having altered protein kinase activity and other variants resistant to growth inhibition by retinoic acid will be selected and the influence of retinoids on the cyclic AMP system in these variants analyzed. Since cyclic AMP-dependent protein kinase is elevated in retinoic acid treated B16 melanoma cells, one can surmise that there could likely be enhanced phosphorylation of cellular proteins. Using the powerful technique of two-dimensional gel electrophoresis, we will identify phosphorylated proteins in control and retinoic acid treated cells and characterize them as to their molecular weight, isoelectric point and subcellular location. One of the exciting areas where retinoids might eventually find therapeutic application is their ability to exert a protective action against chemical carcinogenesis. Since we have obtained evidence that retinoids can effect changes in the cyclic AMP system, the results of the experiments described in this proposal could furnish some insights into ways in which the cyclic AMP system could be manipulated so as to enhance or supplement the action of retinoids.